Mammalian cell culture

ABSTRACT

The invention provides a method for culturing mammalian cells. The method provides greater control over cell growth to achieve high product titer cell cultures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/127,050, filed Apr. 10, 2014; which is a national stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2012/045070, having an international filing date of Jun. 29, 2012and published in English; which claims priority to U.S. ProvisionalPatent Application No. 61/503,737, filed Jul. 1, 2011. The completecontents of foregoing applications are incorporated herein by referencefor all purposes.

FIELD OF INVENTION

The invention provides a method for culturing mammalian cells. Themethod provides greater control over cell growth to achieve high producttiter cell cultures.

BACKGROUND OF INVENTION

As the demand for greater and greater quantities of therapeuticrecombinant proteins increases, positive increases in cell growth,viability and protein production are sought by implementing new methodsto improve cell development, media optimization and process controlparameters. Much effort is now being placed on process optimization,particularly methods and strategies for growing, feeding, andmaintaining production cell cultures.

New cell culture methods that provide even incremental improvements inrecombinant protein production are valuable, given the expense of largescale cell culture processes and the growing demand for greaterquantities of and lower costs for biological products.

Improvements to cell culture processes, recombinant polypeptideexpression, titer, and cell viability that can lead to higher productionlevels, thereby reducing the costs associated with manufacturing proteintherapeutics are needed. The invention fulfills these needs by providinga simple, easy and inexpensive method of controlling cell growth whileincreasing protein production.

SUMMARY OF THE INVENTION

The present invention provides a method of arresting cell growth in amammalian cell culture expressing a recombinant protein comprisingestablishing a mammalian cell culture in a serum-free culture medium ina bioreactor; inducing cell growth-arrest by perfusion with a serum-freeperfusion medium having an L-asparagine concentration of 5 mM or less;maintaining the mammalian cells in a growth-arrested state by perfusionwith a serum-free perfusion medium having an L-asparagine concentrationof 5 mM or less.

The present invention also provides a method of increasing recombinantprotein production in a mammalian cell culture expressing a recombinantprotein comprising establishing a mammalian cell culture in a serum-freeculture medium in a bioreactor; inducing cell growth-arrest by perfusionwith a serum-free perfusion medium having an L-asparagine concentrationof 5 mM or less; maintaining the mammalian cells in a growth-arrestedstate by perfusion with a serum-free perfusion medium having anL-asparagine concentration of 5 mM or less. In a related embodimentrecombinant protein production in the mammalian cell culture isincreased compared to a culture where the cells are not subjected toL-asparagine-induced cell growth-arrest.

The present invention also provides a method of limiting a mammaliancell culture expressing a recombinant protein at a desired packed cellvolume comprising establishing a mammalian cell culture in a serum-freeculture medium in a bioreactor; inducing cell growth-arrest by perfusionwith a serum-free perfusion medium having an L-asparagine concentrationof 5 mM or less; maintaining the mammalian cells in a growth-arrestedstate by perfusion with a serum-free perfusion medium having anL-asparagine concentration of 5 mM or less.

In one embodiment of the present invention, in any of the methods abovethe perfusion with a serum-free perfusion medium having an L-asparagineconcentration of 5 mM or less begins on or before day 3 of the culture.In another embodiment, in any of the methods above induction of cellgrowth-arrest takes place prior to a production phase. In anotherembodiment, in any of the methods above induction of cell growth-arresttakes place during a production phase. In another embodiment, in any ofthe methods above cell growth-arrest is induced by L-asparaginestarvation. In yet another embodiment, any of the methods above furthercomprise a temperature shift from 36° C. to 31° C. In anotherembodiment, any of the methods above further comprise a temperatureshift from 36° C. to 33° C. In a related embodiment the temperatureshift occurs at the transition between a growth phase and a productionphase. In yet another embodiment the temperature shift occurs during aproduction phase. In another embodiment the methods above furthercomprise a packed cell volume during a production phase less than orequal to 35%. In a related embodiment the packed cell volume during aproduction phase is less than or equal to 35%

The present invention also provides a method of culturing mammaliancells expressing a recombinant protein comprising; establishing amammalian cell culture in a serum-free culture medium in a bioreactor;growing the mammalian cells during a growth phase and supplementing theculture medium with bolus feeds of a serum-free feed medium, andmaintaining the mammalian cells during a production phase by perfusionwith a serum-free perfusion medium, wherein the packed cell volumeduring the production phase is less than or equal to 35%. In oneembodiment of the present invention, perfusion begins on or about day 5to on or about day 9 of the cell culture. In a related embodimentperfusion begins on or about day 5 to on or about day 7 of the cellculture. In one embodiment perfusion begins when the cells have reacheda production phase. In another embodiment the method further comprisesinducing cell growth-arrest by L-asparagine starvation followed byperfusion with a serum-free perfusion medium having an L-asparagineconcentration of 5 mM or less. In yet another embodiment the methodfurther comprises inducing cell growth-arrest by perfusion with aserum-free perfusion medium having an L-asparagine concentration of 5 mMor less.

In one embodiment of the invention the concentration of L-asparagine inthe serum-free perfusion medium is less than or equal to 5 mM. Inanother embodiment the concentration of L-asparagine in the serum-freeperfusion medium is less than or equal to 4.0 mM. In another embodimentthe concentration of L-asparagine in the serum-free perfusion medium isless than or equal to 3.0 mM. In still another embodiment theconcentration of L-asparagine in the serum-free perfusion medium is lessthan or equal to 2.0 mM. In yet another embodiment the concentration ofL-asparagine in the serum-free perfusion medium is less than or equal to1.0 mM. In yet another embodiment the concentration of L-asparagine inthe serum-free perfusion medium is 0 mM. In another embodiment perfusionis performed at a rate that increases during the production phase from0.25 working volume per day to 1.0 working volume per day during thecell culture. In a related embodiment perfusion is performed at a ratethat reaches 1.0 working volume per day on day 9 to day 11 of the cellculture. In another related embodiment perfusion is performed at a ratethat reaches 1.0 working volume per day on day 10 of the cell culture.In yet another embodiment the bolus feeds of serum-free feed mediumbegin on day 3 or day 4 of the cell culture. In another embodiment ofthe invention the method further comprises a temperature shift from 36°C. to 31° C. In another embodiment the method further comprises atemperature shift from 36° C. to 33° C. In a related embodiment thetemperature shift occurs at the transition between the growth phase andproduction phase. In a related embodiment the temperature shift occursduring the production phase.

In one embodiment of the invention the L-asparagine concentration of thecell culture medium is monitored prior to and during L-asparaginestarvation.

In one embodiment of the invention the packed cell volume is less thanor equal to 35%. In a related embodiment the packed cell volume is lessthan or equal to 30%.

In one embodiment of the invention the viable cell density of themammalian cell culture at a packed cell volume less than or equal to 35%is 10×10⁶ viable cells/m to 80×10⁶ viable cells/ml. In a relatedembodiment the viable cell density of the mammalian cell culture is20×10⁶ viable cells/ml to 30×10⁶ viable cells/m.

In one embodiment of the invention perfusion comprises continuousperfusion.

In one embodiment of the invention the rate of perfusion is constant.

In one embodiment of the invention perfusion is performed at a rate ofless than or equal to 1.0 working volumes per day.

In another embodiment of the invention the mammalian cell culture isestablished by inoculating the bioreactor with at least 0.5×10⁶ to3.0×10⁶ cells/mL in a serum-free culture medium. In a related embodimentthe mammalian cell culture is established by inoculating the bioreactorwith at least 0.5×10⁶ to 1.5×10⁶ cells/mL in a serum-free culturemedium.

In another embodiment of the invention the perfusion is accomplished byalternating tangential flow.

In another embodiment of the invention the bioreactor has a capacity ofat least 500 L. In a related embodiment the bioreactor has a capacity ofat least 500 L to 2000 L. In yet another related embodiment thebioreactor has a capacity of at least 1000 L to 2000 L.

In another embodiment of the invention the mammalian cells are ChineseHamster

Ovary (CHO)

cells.

In another embodiment of the invention the recombinant protein isselected from the group consisting of a human antibody, a humanizedantibody, a chimeric antibody, a recombinant fusion protein, or acytokine.

In another embodiment of the invention any of the methods above furthercomprise a step of harvesting the recombinant protein produced by thecell culture.

In another embodiment of the invention the recombinant protein producedby the cell culture is purified and formulated in a pharmaceuticallyacceptable formulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Fed-batch start: solid square (▪) and solid circle (•) Batchstart: open square (□) and open circle (∘).

-   -   FIG. 1A: Viable cell density, FIG. 1B: Viability, FIG. 1C: Titer

FIG. 2 Batch start: open circle (∘), Fed-batch start with highagitation: open square (0)

-   -   FIG. 2A Viable cell density, FIG. 2B Viability, FIG. 2C Titer,        FIG. 2D Asparagine concentration

FIG. 3 1.0 start perfusion volume, no temperature shift: solid circle.1.0 start perfusion volume, temperature shift: open circle (∘). 0.75start perfusion volume perfusion volume, no temperature shift: solidsquare (▪). 0.75 start perfusion volume, temperature shift: open square(□).

-   -   FIG. 3A Viable Cell Density, FIG. 3B Viability, FIG. 3C Titer

FIG. 4 Batch start with low asparagine amount: open triangle (Δ). Batchstart with control L-asparagine amount: solid triangle (A). Fed-batchstart with low L-asparagine amount: open diamond (0). Fed-batch startwith control L-asparagine amount: solid diamond (♦). Sparge with drilledpipe: Solid line. Sparge with sintered sparger: dashed line.

-   -   FIG. 4A Viable Cell Density, FIG. 4B Viability, FIG. 4C PCV        Adjusted Titer

FIG. 5 Cultures grown in medium containing 17.3 mM or 5 mM L-asparagineand 4.6 mM or 10 mM L-glutamine. 17.3 mM L-asparagine and 4.6 mML-glutamine solid diamond (♦) or 5 mM L-asparagine, 10 mM L-glutamineopen diamond (0).

-   -   FIG. 5A Viable cell density. FIG. 5B Titer. FIG. 5C Packed cell        volume (PCV). FIG. 5D PCV-adjusted titer. FIG. 5E Viability.

FIG. 6 . Cultures at 2 L bench scale and 500 L pilot scale, with 5 mML-asparagine, 10 mM L-glutamine. Medium containing 5 mM L-asparagine, 10mM L-glutamine at 2 L bench scale is represented by the solid diamond(♦) and the 500 L pilot scale is represented by the open diamond (0)

-   -   FIG. 6A Viable cell density. FIG. 6B Titer. FIG. 6C Packed cell        volume (PCV). FIG. 6D PCV-adjusted titer. FIG. 6E Viability.

DETAILED DESCRIPTION OF THE INVENTION

During recombinant protein production it is desirable to have acontrolled system where cells are grown to a desired density and thenthe physiological state of the cells is switched to a growth-arrested,high productivity state where the cells use energy and substrates toproduce the recombinant protein of interest instead of making morecells. Methods for accomplishing this goal, such as temperature shiftsand small molecule inducers, are not always successful and can haveundesirable effects on product quality. As described herein, packed cellvolume can be limited to a desired level during the production phase byinducing cell growth-arrest in the cultured cells by exposure to lowL-asparagine conditions. Cell growth-arrest can be achieved andmaintained by using a perfusion culture medium that contains a limitingconcentration of L-asparagine and maintaining a low concentration ofL-asparagine in the cell culture (5 mM or less).

It was also found that the growth-arrested cells showed increasedproductivity when growth-arrest was initiated by low L-asparagine orthrough L-asparagine starvation and the growth-arrested cells weresubsequently maintained with the cell culture and perfusion mediumhaving an L-asparagine concentration of 5 mM or less.

A growth-arrested, high productivity production phase can be achieved bymanipulating the concentration of L-asparagine. As described herein,depletion of L-asparagine resulted in growth-arrest. In a fed-batchculture, once the cell density was sufficiently high (for example>20×10⁶ viable cells/mL), the culture was repeatedly starved ofL-asparagine despite repeated feedings due to consumption ofL-asparagine and/or the conversion to L-aspartate. In a cell culture,the extracellular L-asparagine can be converted to L-aspartate andammonia. L-asparagine depletion resulted in cell cycle arrest. Duringfed-batch, periods when L-asparagine is present in the culture result inincreased productivity and periods when L-asparagine has been depletedresult in decreased productivity. In the perfused system, L-asparagineis constantly supplied, total depletion is therefor avoided, and higherconcentrations of L-asparagine can be sustained, thus allowing cells tocontinue multiplying and not be exposed to and environment with depletedor limited L-asparagine. Controlling the concentration of L-asparagineat a sufficiently low concentration (such as concentrations 5 mM orless) can keep the cells in a high productivity state while maintainingviability and limiting growth. In a system having bolus and perfusionfeeding, the feed medium can be switched from a formulation containing ahigh (growth promoting) level of L-asparagine during bolus feeds to alower (growth-arresting) level of L-asparagine during perfusion feeding.Cell cultures that have been growth-arrested by limiting L-asparaginecan be stimulated into a high productivity state by adding back lowlevels of L-asparagine.

For commercial scale cell culture and the manufacture of biologicaltherapeutics, the ability to arrest cell growth and being able tomaintain the cells in a growth-arrested state during the productionphase would be very desirable. Having cells that were also induced toincrease productivity while in the growth-arrested state and being ableto maintain this increased productivity, is ideal for manufacturingpurposes.

Provided herein is a method of arresting cell growth in a mammalian cellculture expressing a recombinant protein. The method includes inducingcell growth-arrest in a mammalian cell culture by subjecting the cellculture to serum-free medium having an L-asparagine concentration of 5mM or less which includes OmM L-asparageine). Such induction can beinitiated by L-asparagine starvation or by creating a low L-asparagineenvironment by perfusing the culture with a serum-free perfusion mediumhaving an L-asparagine concentration of 5 mM or less and maintaining theculture in a low L-asparagine environment. The cell culture is thenmaintained in the growth-arrested state by perfusing with a serum-freeperfusion medium with an L-asparagine at a concentration of 5 mM or lessand maintaining the culture in the low L-asparagine environment.

Also provided is a method for increasing recombinant protein productionin a mammalian cell culture expressing a recombinant protein by inducinga low asparagine cell growth-arrest in a mammalian cell culture.Mammalian cells maintained in the low asparagine growth-arrested stateexhibited greater productivity (g protein/cell/day and g protein/cellmass/day) than those which were not low asparagine growth-arrested.

Such method is also useful for limiting a mammalian cell culture at adesired packed cell volume. Packed cell volume during the productionphase could be limited at a desired level by reducing L-asparaginelevels in the production culture medium. Asparagine concentrations of 5mM or less in the perfusion medium were sufficient to control cellgrowth during culture and limit to a desired packed cell volume.

The methods described herein provide greater control over cell growth tohigh product titer cell cultures; and as such can simplify the gassingstrategy compared to a high biomass perfusion processes and minimizeproduct loss during harvest and downstream processing.

The method begins with establishing a mammalian cell culture in aproduction bioreactor. Preferably smaller production bioreactors areused, in one embodiment the bioreactors are 500 L to 2000 L. In apreferred embodiment, 1000 L-2000 L bioreactors are used. The seed celldensity used to inoculate the bioreactor can have a positive impact onthe level of recombinant protein produced. In one embodiment thebioreactor is inoculated with at least 0.5×lO⁶ up to and beyond 3.0×lO⁶viable cells/mL in a serum-free culture medium. In a preferredembodiment the inoculation is l.O×lO⁶ viable cells/mL.

The mammalian cells then undergo an exponential growth phase. The cellculture can be maintained without supplemental feeding until a desiredcell density is achieved. In one embodiment the cell culture ismaintained for up to 3 days without supplemental feeding followed byperfusion with a serum-free perfusion medium having an L-asparagineconcentration of 5 mM or less to induce and maintain low L-asparaginegrowth-arrest. In another embodiment the culture can be inoculated at adesired cell density to begin the production phase without a briefgrowth phase with cell growth-arrest initiated immediately uponinoculation by perfusing the cell culture with serum-free perfusionmedium containing 5 mM or less L-asparagine to induce and maintain lowL-asparagine growth-arrest. In any of the embodiments herein the switchfrom the growth phase to production phase can also be initiated byL-asparagine starvation (subjecting cells to a 0 mM L-asparagineenvironment) followed by perfusion with a cell culture medium having anL-asparagine concentration of equal to or less than 5 mM, andmaintaining the concentrating of L-asparagine in the cell culture atthat level.

Without regard as to how low L-asparagine growth-arrest is induced,higher productivity is seen in the growth-arrested cells that aremaintained by perfusing with a low L-asparagine medium and maintainingthe cell culture at an L-asparagine level of 5 mM or less.

As used herein, “growth-arrest”, which may also be referred to as “cellgrowth-arrest”, is the point where cells stop increasing in number orwhen the cell cycle no longer progresses. Growth-arrest can be monitoredby determining the viable cell density of a cell culture. Some cells ina growth-arrested state may increase in size but not number, so thepacked cell volume of a growth-arrested culture may increase.Growth-arrest can be reversed to some extent, if the cells are not indeclining health, by adding additional L-asparagine to the cell culture.

Growth-arrest is initiated by L-asparagine when the cell density of theculture reaches a level where the concentration of L-asparagine in theculture becomes limiting for continued growth or when the culture isstarved of L-asparagine. L-asparagine starvation occurs when theL-asparagine concentration in a cell culture medium is effectively atOmM. Starvation can result in growth-arrest within 24 hours. Starvationfor longer than 48 hours could damage the health of the cells. Tomaintain cells in the growth-arrested state, the L-asparagineconcentration in the cell culture must be maintained at 5 mM or less.The cell culture medium concentration of L-asparagine required to arrestcell growth is dependent on the ability of the cells to make their ownasparagine. For cultures where cells can make their own asparagine, alower concentration, or even removal of L-asparagine from the medium maybe required for growth-arrest. For cultures that are unable to maketheir own asparagine, for example, cells that lack active asparaginesynthetase enzyme, concentrations above zero up to 5 mM L-asparaginecould be used to arrest growth.

As used herein, “packed cell volume” (PCV), also referred to as “percentpacked cell volume” (% PCV), is the ratio of the volume occupied by thecells, to the total volume of cell culture, expressed as a percentage(see Stettler, wt al., (2006) Biotechnol Bioeng. Dec 20:95(6): 1228-33).Packed cell volume is a function of cell density and cell diameter;increases in packed cell volume could arise from increases in eithercell density or cell diameter or both. Packed cell volume is a measureof the solid content in the cell culture. Solids are removed duringharvest and downstream purification. More solids mean more effort toseparate the solid material from the desired product during harvest anddownstream purification steps. Also, the desired product can becometrapped in the solids and lost during the harvest process, resulting ina decreased product yield. Since host cells vary in size and cellcultures also contain dead and dying cells and other cellular debris,packed cell volume is a more accurate way to describe the solid contentwithin a cell culture than cell density or viable cell density. Forexample, a 2000 L culture having a cell density of 50×10⁶ cells/m wouldhave vastly different packed cell volumes depending on the size of thecells. In addition, some cells, when in a growth-arrested state, willincrease in size, so the packed cell volume prior to growth-arrest andpost growth-arrest will likely be different, due to increase in biomassas a result to cell size increase.

At the transition between the growth phase and the production phase, andduring the production phase, the percent packed cell volume (% PCV) isequal to or less than 35%. The desired packed cell volume maintainedduring the production phase is equal to or less than 35%. In a preferredembodiment the packed cell volume is equal to or less than 30%. Inanother preferred embodiment the packed cell volume is equal to or lessthan 20%. In another preferred embodiment the packed cell volume isequal to or less than 15%. In yet another preferred embodiment thepacked cell volume is equal to or less than 10%.

As used herein, “cell density” refers to the number of cells in a givenvolume of culture medium. “Viable cell density” refers to the number oflive cells in a given volume of culture medium, as determined bystandard viability assays (such as trypan blue dye exclusion method).

The desired viable cell density at the transition between the growth andproduction phases and maintained during the production phase is one thatprovides a packed cell volume of equal to or less than 35%. In oneembodiment, the viable cell density is at least about lO×lO⁶ viablecells/mL to 80×10⁶ viable cells/mL. In one embodiment the viable celldensity is at least about lO×lO⁶ viable cells/mL to 70×10⁶ viablecells/mL. In one embodiment the viable cell density is at least aboutlO×lO⁶ viable cells/mL to 60×1 0⁶ viable cells/mL. In one embodiment theviable cell density is at least about lO×lO⁶ viable cells/mL to 50×10⁶viable cells/mL. In one embodiment the viable cell density is at leastabout lO×lO⁶ viable cells/mL to 40×10⁶ viable cells/mL. In a preferredembodiment the viable cell density is at least about lO×lO⁶ viablecells/mL to 30×10⁶ viable cells/mL. In another preferred embodiment theviable cell density is at least about lO×lO⁶ viable cells/mL to 20×10⁶viable cells/mL. In another preferred embodiment, the viable celldensity is at least about 20×10⁶ viable cells/mL to 30×10⁶ viablecells/mL. In another preferred embodiment the viable cell density is atleast about 20×10⁶ viable cells/mL to at least about 25×10⁶ viablecells/mL, more preferably at least about 20×10⁶ viable cells/mL.

Lower packed cell volume during the production phase helps mitigatedissolved oxygen sparging problems that can hinder higher cell densityperfusion cultures. The lower packed cell volume also allows for asmaller media volume which allows for the use of smaller media storagevessels and can be combined with slower flow rates. Lower packed cellvolume also has less impact on harvest and downstream processing,compared to higher cell biomass cultures. All of which reduces the costsassociated with manufacturing recombinant protein therapeutics.

Three methods are typically used in commercial processes for theproduction of recombinant proteins by mammalian cell culture: batchculture, fed-batch culture, and perfusion culture. Batch culture, adiscontinuous method where cells are grown in a fixed volume of culturemedia for a short period of time followed by a full harvest. Culturesgrown using the batch method experience an increase in cell densityuntil a maximum cell density is reached, followed by a decline in viablecell density as the media components are consumed and levels ofmetabolic by-products (such as lactate and ammonia) accumulate. Harvesttypically occurs at the point when the maximum cell density is achieved(typically 5-10×10⁶ cells/mL, depending on media formulation, cell line,etc). The batch process is the simplest culture method, however viablecell density is limited by the nutrient availability and once the cellsare at maximum density, the culture declines and production decreases.There is no ability to extend a production phase because theaccumulation of waste products and nutrient depletion rapidly lead toculture decline, (typically around 3 to 7 days).

Fed-batch culture improves on the batch process by providing bolus orcontinuous media feeds to replenish those media components that havebeen consumed. Since fed-batch cultures receive additional nutrientsthroughout the run, they have the potential to achieve higher celldensities (>10 to 30×10⁶ cells/ml, depending on media formulation, cellline, etc)) and increased product titers, when compared to the batchmethod. Unlike the batch process, a biphasic culture can be created andsustained by manipulating feeding strategies and media formulations todistinguish the period of cell proliferation to achieve a desired celldensity (the growth phase) from the period of suspended or slow cellgrowth (the production phase). As such, fed batch cultures have thepotential to achieve higher product titers compared to batch cultures.Typically a batch method is used during the growth phase and a fed-batchmethod used during the production phase, but a fed-batch feedingstrategy can be used throughout the entire process. However, unlike thebatch process, bioreactor volume is a limiting factor which limits theamount of feed. Also, as with the batch method, metabolic by-productaccumulation will lead to culture decline, which limits the duration ofthe production phase, about 1.5 to 3 weeks. Fed-batch cultures arediscontinuous and harvest typically occurs when metabolic by-productlevels or culture viability reach predetermined levels.

Perfusion methods offer potential improvement over the batch andfed-batch methods by adding fresh media and simultaneously removingspent media. Typical large scale commercial cell culture strategiesstrive to reach high cell densities, 60-90(+)×10⁶ cells/mL where almosta third to over one-half of the reactor volume is biomass. Withperfusion culture, extreme cell densities of >1×10⁸ cells/mL have beenachieved and even higher densities are predicted. Typical perfusioncultures begin with a batch culture start-up lasting for a day or twofollowed by continuous, step-wise and/or intermittent addition of freshfeed media to the culture and simultaneous removal of spend media withthe retention of cells and additional high molecular weight compoundssuch as proteins (based on the filter molecular weight cutoff)throughout the growth and production phases of the culture. Variousmethods, such as sedimentation, centrifugation, or filtration, can beused to remove spent media, while maintaining cell density. Perfusionflow rates of a fraction of a working volume per day up to many multipleworking volumes per day have been reported. An advantage of theperfusion process is that the production culture can be maintained forlonger periods than batch or fed-batch culture methods. However,increased media preparation, use, storage and disposal are necessary tosupport a long term perfusion culture, particularly those with high celldensities, which also need even more nutrients, and all of this drivesthe production costs even higher, compared to batch and fed batchmethods. In addition, higher cell densities can cause problems duringproduction, such as maintaining dissolved oxygen levels and problemswith increased gassing including supplying more oxygen and removing morecarbon dioxide, which would result in more foaming and the need foralterations to antifoam strategies; as well as during harvest anddownstream processing where the efforts required to remove the excessivecell material can result in loss of product, negating the benefit ofincreased titer due to increased cell mass.

Also provided is a large scale cell culture strategy that combines fedbatch feeding during the growth phase followed by continuous perfusionduring the production phase. The method targets a production phase wherethe cell culture is maintained at a packed cell volume of less than orequal to 35%. The method also provides the initiation and maintenance ofcell growth-arrest due to low asparagine.

Fed batch culture is a widely-practiced culture method for large scaleproduction of proteins from mammalian cells. See e.g. Chu and Robinson(2001), Current Opin. Biotechnol. 12: 180-87. A fed batch culture ofmammalian cells is one in which the culture is fed, either continuouslyor periodically, with a concentrated feed medium containing nutrients.Feeding can occur on a predetermined schedule of, for example, everyday, once every two days, once every three days, etc. When compared to abatch culture, in which no feeding occurs, a fed batch culture canproduce greater amounts of recombinant protein. See e.g. U.S. Pat. No.5,672,502.

In one embodiment, a fed-batch culture with bolus feeds is used tomaintain a cell culture during the growth phase. Perfusion feeding canthen be used during a production phase. In one embodiment, perfusionbegins when the cells have reached a production phase. In anotherembodiment, perfusion begins on or about day 5 to on or about day 9 ofthe cell culture. In another embodiment perfusion begins on or about day5 to on or about day 7 of the cell culture.

In another embodiment the initiation of cell growth-arrest in thefed-batch culture can be initiated by subjecting the fed-batch cultureto a period of L-asparagine starvation followed by perfusion with aserum-free perfusion medium having an L-asparagine concentration of 5 mMor less. In one embodiment the L-asparagine concentration of the cellculture medium is monitored prior to and during L-asparagine starvation.In another embodiment the initiation of cell growth-arrest in thefed-batch culture can be achieved by perfusion with a serum freeperfusion medium having an L-asparagine concentration of 5 mM or less.

Using bolus feeding during the growth phase allows the cells totransition into the production phase, resulting in less dependence on atemperature shift as a means of initiating and controlling theproduction phase, however a temperature shift of 36° C. to 31° C. cantake place between the growth phase and production phase. In a preferredembodiment the shift is from 36° C. to 33° C.

As described herein, the bioreactor can be inoculated with at least0.5×10⁶ up to and beyond 3.0×10⁶ viable cells/mL in a serum-free culturemedium, preferably 1.0×10⁶ viable cells/mL.

Perfusion culture is one in which the cell culture receives freshperfusion feed medium while simultaneously removing spent medium.Perfusion can be continuous, step-wise, intermittent, or a combinationof any or all of any of these. Perfusion rates can be less than aworking volume to many working volumes per day. Preferably the cells areretained in the culture and the spent medium that is removed issubstantially free of cells or has significantly fewer cells than theculture. Recombinant proteins expressed by the cell culture can also beretained in the culture. Perfusion can be accomplished by a number ofmeans including centrifugation, sedimentation, or filtration, See e.g.Voisard et al., (2003), Biotechnology and Bioengineering 82:751-65. Apreferred filtration method is alternating tangential flow filtration.Alternating tangential flow is maintained by pumping medium throughhollow-fiber filter modules. See e.g. U.S. Pat. No. 6,544,424; Furey(2002) Gen. Eng. News. 22 (7). 62-63.

As used herein, “perfusion flow rate” is the amount of media that ispassed through (added and removed) from a bioreactor, typicallyexpressed as some portion or multiple of the working volume, in a giventime. “Working volume” refers to the amount of bioreactor volume usedfor cell culture. In one embodiment the perfusion flow rate is oneworking volume or less per day. Perfusion feed medium can be formulatedto maximize perfusion nutrient concentration to minimize perfusion rate.

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells may be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, can be used. In oneembodiment 500 L to 2000 L bioreactors are used. In a preferredembodiment, 1000 L to 2000 L bioreactors are used.

For the purposes of this invention, cell culture medium is a mediasuitable for growth of animal cells, such as mammalian cells, in invitro cell culture. Cell culture media formulations are well known inthe art. Typically, cell culture media are comprised of buffers, salts,carbohydrates, amino acids, vitamins and trace essential elements.“Serum-free” applies to a cell culture medium that does not containanimal sera, such as fetal bovine serum. Various tissue culture media,including defined culture media, are commercially available, forexample, any one or a combination of the following cell culture mediacan be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's ModifiedEagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium.Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's SAMedium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™300 Series (JRH Biosciences, Lenexa, Kans.), among others. Serum-freeversions of such culture media are also available. Cell culture mediamay be supplemented with additional or increased concentrations ofcomponents such as amino acids, salts, sugars, vitamins, hormones,growth factors, buffers, antibiotics, lipids, trace elements and thelike, depending on the requirements of the cells to be cultured and/orthe desired cell culture parameters.

Cell cultures can be supplemented with concentrated feed mediumcontaining components, such as nutrients and amino acids, which areconsumed during the course of the production phase of the cell culture.Concentrated feed medium may be based on just about any cell culturemedia formulation. Such a concentrated feed medium can contain most ofthe components of the cell culture medium at, for example, about 5×, 6×,7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, lOO×, 200×, 400×, 600×,800×, or even about 1000× of their normal amount. Concentrated feedmedia are often used in fed batch culture processes.

The method according to the present invention may be used to improve theproduction of recombinant proteins in multiple phase culture processes.In a multiple stage process, cells are cultured in two or more distinctphases. For example cells may be cultured first in one or more growthphases, under environmental conditions that maximize cell proliferationand viability, then transferred to a production phase, under conditionsthat maximize protein production. In a commercial process for productionof a protein by mammalian cells, there are commonly multiple, forexample, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases thatoccur in different culture vessels preceding a final production culture.The growth and production phases may be preceded by, or separated by,one or more transition phases. In multiple phase processes, the methodaccording to the present invention can be employed at least during thegrowth and production phase of the final production phase of acommercial cell culture, although it may also be employed in a precedinggrowth phase. A production phase can be conducted at large scale. Alarge scale process can be conducted in a volume of at least about 100,500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15.000, 20,000 liters.In a preferred embodiment production is conducted in 500 L, 1000 Land/or 2000 L bioreactors. A growth phase may occur at a highertemperature than a production phase. For example, a growth phase mayoccur at a first temperature from about 35° C. to about 38° C., and aproduction phase may occur at a second temperature from about 29° C. toabout 37° C., optionally from about 30° C. to about 36° C. or from about30° C. to about 34° C. In addition, chemical inducers of proteinproduction, such as, for example, caffeine, butyrate, and hexamethylenebisacetamide (HMBA), may be added at the same time as, before, and/orafter a temperature shift. If inducers are added after a temperatureshift, they can be added from one hour to five days after thetemperature shift, optionally from one to two days after the temperatureshift. The cell cultures can be maintained for days or even weeks whilethe cells produce the desired protein(s).

Samples from the cell culture can be monitored and evaluated using anyof the analytical techniques known in the art. Variety of parametersincluding recombinant protein and medium quality and characteristics canbe monitored for the duration of the culture. Samples can be taken andmonitored intermittently at a desirable frequency, including continuousmonitoring, real time or near real time. In one embodiment theL-asparagine concentration of the cell culture medium is monitored priorto and during L-asparagine starvation.

Typically the cell cultures that precede the final production culture(N−x to N−1) are used to generate the seed cells that will be used toinoculate the production bioreactor, the N−1 culture. The seed celldensity can have a positive impact on the level of recombinant proteinproduced. Product levels tend to increase with increasing seed density.Improvement in titer is tied not only to higher seed density, but islikely to be influenced by the metabolic and cell cycle state of thecells that are placed into production.

Seed cells can be produced by any culture method. A preferred method isa perfusion culture using alternating tangential flow filtration. An N−1bioreactor can be run using alternating tangential flow filtration toprovide cells at high density to inoculate a production bioreactor. TheN−1 stage may be used to grow cells to densities of >90×10⁶ cells/mL.The N−1 bioreactor can be used to generate bolus seed cultures or can beused as a rolling seed stock culture that could be maintained to seedmultiple production bioreactors at high seed cell density. The durationof the growth stage of production can range from 7 to 14 days and can bedesigned so as to maintain cells in exponential growth prior toinoculation of the production bioreactor. Perfusion rates, mediumformulation and timing are optimized to grow cells and deliver them tothe production bioreactor in a state that is most conducive tooptimizing their production. Seed cell densities of >15×10⁶ cells/mL canbe achieved for seeding production bioreactors. Higher seed celldensities at inoculation can decrease or even eliminate the time neededto reach a desired production density.

The invention finds particular utility in improving cell growth,viability and/or protein production via cell culture processes. The celllines (also referred to as “host cells”) used in the invention aregenetically engineered to express a polypeptide of commercial orscientific interest. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. Genetically engineering the cell line involvestransfecting, transforming or transducing the cells with a recombinantpolynucleotide molecule, and/or otherwise altering (e.g., by homologousrecombination and gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express a desiredrecombinant polypeptide. Methods and vectors for genetically engineeringcells and/or cell lines to express a polypeptide of interest are wellknown to those of skill in the art, for example, various techniques areillustrated in Current Protocols in Molecular Biology, Ausubel et al.,eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring LaboratoryPress, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990,pp. 15-69.

Animal cell lines are derived from cells whose progenitors were derivedfrom a multicellular animal. One type of animal cell line is a mammaliancell line. A wide variety of mammalian cell lines suitable for growth inculture are available from the American Type Culture Collection(Manassas, Va.) and commercial vendors. Examples of cell lines commonlyused in the industry include VERO, BHK, HeLa, CV (including Cos), MDCK,293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, andChinese hamster ovary (CHO) cells. CHO cells are widely used for theproduction of complex recombinant proteins, e.g. cytokines, clottingfactors, and antibodies (Brasel et al. (1996), Blood 88:2004-2012;Kaufman et al. (1988), J. Biol Chem 263:6352-6362; McKinnon et al.(1991), J Mol Endocrinol 6:231-239; Wood et al. (1990), J. Immunol.145:3011-3016). The dihydrofolate reductase (DHFR)-deficient mutant celllines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the efficientDHFR selectable and amplifiable gene expression system allows high levelrecombinant protein expression in these cells (Kaufman R. J. (1990),Meth Enzymol 185:537-566). In addition, these cells are easy tomanipulate as adherent or suspension cultures and exhibit relativelygood genetic stability. CHO cells and proteins recombinantly expressedin them have been extensively characterized and have been approved foruse in clinical commercial manufacturing by regulatory agencies.

The methods of the invention can be used to culture cells that expressrecombinant proteins of interest. The expressed recombinant proteins maybe secreted into the culture medium from which they can be recoveredand/or collected. In addition, the proteins can be purified, orpartially purified, from such culture or component (e.g., from culturemedium) using known processes and products available from commercialvendors. The purified proteins can then be “formulated”, meaning bufferexchanged, sterilized, bulk-packaged, and/or packaged for a final user.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 18th ed. 1995, MackPublishing Company, Easton, Pa.

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation. Polypeptides can be of scientific or commercialinterest, including protein-based drugs. Polypeptides include, amongother things, antibodies, fusion proteins, and cytokines. Peptides,polypeptides and proteins are produced by recombinant animal cell linesusing cell culture methods and may be referred to as “recombinantpeptide”, “recombinant polypeptide” and “recombinant protein”. Theexpressed protein(s) may be produced intracellularly or secreted intothe culture medium from which it can be recovered and/or collected.

Examples of polypeptides that can be produced with the methods of theinvention include proteins comprising amino acid sequences identical toor substantially similar to all or part of one of the followingproteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391),erythropoeitin, thrombopoeitin, calcitonin, IL-2, angiopoietin-2(Maisonpierre et al. (1997), Science 277(5322): 55-60), ligand forreceptor activator of NF-kappa B (RANKL, WO 01/36637), tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633),thymic stroma-derived lymphopoietin, granulocyte colony stimulatingfactor, granulocyte-macrophage colony stimulating factor (GM-CSF,Australian Patent No. 588819), mast cell growth factor, stem cell growthfactor (U.S. Pat. No. 6,204,363), epidermal growth factor, keratinocytegrowth factor, megakaryote growth and development factor, RANTES, humanfibrinogen-like 2 protein (FGL2; NCBI accession no. NM_00682; Ruegg andPytela (1995), Gene 160:257-62) growth hormone, insulin, insulinotropin,insulin-like growth factors, parathyroid hormone, interferons includingα-interferons, γ-interferon, and consensus interferons (U.S. Pat. Nos.4,695,623 and 4,897,471), nerve growth factor, brain-derivedneurotrophic factor, synaptotagmin-like proteins (SLP 1-5),neurotrophin-3, glucagon, interleukins, colony stimulating factors,lymphotoxin-β, leukemia inhibitory factor, and oncostatin-M.Descriptions of proteins that can be produced according to the inventivemethods may be found in, for example, Human Cytokines: Handbook forBasic and Clinical Research, all volumes (Aggarwal and Gutterman, eds.Blackwell Sciences, Cambridge, Mass., 1998); Growth Factors: A PracticalApproach (McKay and Leigh, eds., Oxford University Press Inc., New York,1993); and The Cytokine Handbook. Vols. 1 and 2 (Thompson and Lotzeeds., Academic Press, San Diego, Calif., 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. Nos. 5,395,760 and5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EP PatentNo. 0460846, U.S. Pat. Nos. 4,968,607, and 5,767,064), IL-1 receptorantagonists (U.S. Pat. No. 6,337,072), IL-1 antagonists or inhibitors(U.S. Pat. Nos. 5,981,713, 6,096,728, and 5,075,222) IL-2 receptors,IL-4 receptors (EP Patent No. 0 367 566 and U.S. Pat. No. 5,856,296),IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No.6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and4), and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the Vlth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 19%). SimilarCD proteins are disclosed in subsequent workshops. Examples of suchantigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto (CD27ligand, CD30 ligand, etc.). Several of the CD antigens are members ofthe TNF receptor family, which also includes 4IBB and OX40. The ligandsare often members of the TNF family, as are 4IBB ligand and OX40 ligand.

Enzymatically active proteins or their ligands can also be producedusing the invention. Examples include proteins comprising all or part ofone of the following proteins or their ligands or a proteinsubstantially similar to one of these: a disintegrin andmetalloproteinase domain family members including TNF-alpha ConvertingEnzyme, various kinases, glucocerebrosidase, superoxide dismutase,tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The term “antibody” includes reference to both glycosylated andnon-glycosylated immunoglobulins of any isotype or subclass or to anantigen-binding region thereof that competes with the intact antibodyfor specific binding, unless otherwise specified, including human,humanized, chimeric, multi-specific, monoclonal, polyclonal, andoligomers or antigen binding fragments thereof. Also included areproteins having an antigen binding fragment or region such as Fab, Fab′,F(ab′)2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibodymolecules, complementarity determining region (CDR) fragments, scFv,diabodies, triabodies, tetrabodies and polypeptides that contain atleast a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to a target polypeptide. The term “antibody” isinclusive of, but not limited to, those that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those thatrecognize any one or a combination of proteins including, but notlimited to, the above-mentioned proteins and/or the following antigens:CD2, CD3, CD4, CD8, CDl 1a, CD14, CD18, CD20, CD22, CD23, CD25, CD33,CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1a, IL-1β, IL-2,IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF,TGF-β2, TGF-βI, EGF receptor (see U.S. Pat. No. 6,235,883) VEGFreceptor, hepatocyte growth factor, osteoprotegerin ligand, interferongamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1,and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigenMUC, PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, HER-3, a tumor-associatedglycoprotein TAG-72, the SK-1 antigen, tumor-associated epitopes thatare present in elevated levels in the sera of patients with colon and/orpancreatic cancer, cancer-associated epitopes or proteins expressed onbreast, colon, squamous cell, prostate, pancreatic, lung, and/or kidneycancer cells and/or on melanoma, glioma, or neuroblastoma cells, thenecrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4,B2 integrins, TRAIL receptors 1, 2, 3, and 4. RANK. RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor Vila-tissuefactor), MHC 1, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-I receptor, HLA-DR 10 beta, HLA-DRantigen, sclerostin, L-selectin, Respiratory Syncitial Virus, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcusmutans, and Staphlycoccus aureus. Specific examples of known antibodieswhich can be produced using the methods of the invention include but arenot limited to adalimumab, bevacizumab, infliximab, abciximab,alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan,labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

The invention can also be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Hakansson et al. (1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a receptor is fused to an Fc portionof an antibody such as etanercept (a p75 TNFR:Fc), and belatacept(CTLA4:Fc).

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. The methods andtechniques described herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990). All documents, or portions of documents, cited inthis application, including but not limited to patents, patentapplications, articles, books, and treatises, are hereby expresslyincorporated by reference. What is described in an embodiment of theinvention can be combined with other embodiments of the invention.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention.

Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

EXAMPLES Example 1

This experiment compares different start up conditions using batch orfed batch feeding methods followed by continuous perfusion usingalternating tangential flow filtration. Perfusion was started eitherearly during the early exponential growth phase (“batch” start up) withno additional feeds prior to perfusion or at the end of the exponentialphase and entering into the stationary or production phase (“fed-batch”start up) receiving several bolus feeds of a serum-free defined feedmedia prior to perfusion.

On day 0, CHO cells expressing a recombinant antibody were inoculatedinto 2 L production bioreactors at l×lO⁶ viable cells/mL in a workingvolume of 1500 ml of a serum-free defined medium for the fed-batch startand 1800 mL for the batch start. Cultures were maintained at 36° C.,dissolved oxygen (DO) at 30%, agitation at 215 RPM. Glucose wasmaintained above 0 g/L and below 8 g/L.

Perfusion was started on day 4 (0.25 Vol/day) for the batch cultures andon day 7 (0.75 Vol/day) for the fed-batch cultures. Perfusion wasaccomplished using an alternating tangential flow perfusion andfiltration system (Refine Technologies, Hanover, N.J., 50 kDa hollowfiber filter). Prior to starting perfusion the fed-batch culturesreceived bolus feeds of a concentrated serum-free defined feed media onday 4 (7.5% of initial working volume) and day 6 (10% initial workingvolume). Perfusion rates are provided in Table 1.

TABLE 1 Perfusion Rate Perfusion Rate Day (Vol/day)  0-4* 0.00  4-6 0.25 6-7 0.50  7-10 0.75 10- 1.00 Values are based on working volumesdisclosed above *Day 0-7 for the fed batch start

During the culture run, daily samples were taken to assess the culture.Viable cell density (VCD) and viability were determined using Vi-Cell(Beckman Coulter, Brea, Calif.). Titer was measured by HPLC analysis.Packed cell volume was determined using VoluPAC (Sartorius, Goettingen,Germany).

A temperature shift (36.0° C. to 33.0° C.) was applied when the viablecell density exceeded 20×10⁶ viable cells/mL which was day 7 and day 11for the batch and fed-batch start conditions respectively.

For the batch start-up conditions, the viable cell density continued toincrease after the perfusion was started; for the fed-batch startconditions, the perfusion was started after the cell culture had reacheda plateau or stationary phase with little growth. On day 15, the viablecell density for the fed batch was between 27.7 and 30.7×10⁶ viablecells/mL while VCD of the batch culture was between 22.5 and 27.4×10⁶viable cells/mL (FIG. 1A). Viability of the fed batch culture wasbetween 73.9 and 77.5% while the batch culture viability was between72.5 and 83.1% (FIG. 1B). The titer of the fed batch culture was between15.3 and 16.1 g/L while the batch culture titer was between 10.6 and12.3 g/L (FIG. 1C). Since the integrated variable cell density (IVCD)values were similar for all four cultures by day 15 (approximately230×10⁶ cell days/mL), the specific productivity was higher in thefed-batch start-up conditions. The fed batch cultures were continued today 24. A titer of 20 g/L was achieved at 20 days.

The alternating tangential flow perfusion with a fed batch start-upresulted in increased productivity, maintaining the cells in a moreproductive state compared to the batch start method.

Example 2

On day 0, CHO cells expressing a recombinant antibody were inoculatedinto 2 L production bioreactors at l×lO⁶ viable cells/mL in a workingvolume of 1500 ml of a serum-free defined medium for the batch start and1300 mL for the fed-batch start. Cultures were maintained at 36° C., DOat 30%, agitation at 215 RPM for batch cultures. The fed-batch culturewas agitated at 430 RPM. Fed-batch culture was fed to 7 g/L glucosedaily prior to perfusion and all cultures were maintained at or above 4g/L glucose during perfusion. Perfusion (alternating tangential flow)was started on day 4 (0.25 Vol/day) for the batch cultures and on day 8(0.75 Vol/day) for the fed-batch culture. Prior to starting perfusionthe fed-batch culture received bolus feeds of a concentrated serum-freedefined feed media on day 4 (7.5% of initial working volume) and day 6(10% initial working volume). Perfusion flow rates settings are providedin Table 2. The cultures were maintained for 21 days.

TABLE 2 Perfusion Rate Perfusion Rate Day (Vol/day)   4-6 0.25   6-80.50   8-10 0.75  10- 1.00 Values are based on working volumes disclosedabove

During the culture run daily samples were taken as described above toassess the culture.

A temperature shift (36.0° C. to 33.0° C.) was applied to the batchcultures on day 6 when the viable cell density exceeded 20×10⁶ viablecells/mL, as in Example 1 The fed batch culture was maintained at 36.0°C. for the duration of the culture.

The batch start-up method cultures had results similar to thosedescribed above with the cells reaching approximately 20 to 25×10⁶viable cells/mL with no growth after day 10. The fed-batch culturereached almost 30×10⁶ viable cells/mL on day 20 after spending most ofthe culture duration below 20×10⁶ viable cells/mL, see FIG. 2A. Theviabilities all remained above 80% until day 10 and then dropping toabout 40% by day 20 for the batch start cultures and 60% for thefed-batch culture, see FIG. 2B. The titer peaked at almost 15 g/L forthe batch start cultures, but it reached over 20 g/L for the highagitation fed-batch culture, see FIG. 2C. It was observed that the batchstart cultures had an L-asparagine concentration of about 3 to 4 mM onday 3, and did not experience an asparagine limited culture environment.However, the fed-batch perfusion start culture experienced anL-asparagine limited environment by day 6 prior to the start of theperfusion on day 7. The culture was then perfused with medium containingL-asparagine at a concentration of 2.0 g/L (or 13.3 mM) that resulted inno further L-asparagine limitations after day 8 (FIG. 2D). Glucoseconcentrations were maintained mostly between 4 and 10 g/L.

The fed-batch start-up with high agitation perfusion culture achievedthe highest titer (over 20 g/L) in 20 days, more than 5 g/L higher thanthe batch start-up cultures, which was similar to the results describedabove. No negative effects of a higher agitation rate were observed.Maintaining a constant temperature did not appear to negatively impactthe fed batch culture.

Example 3

This experiment characterizes the effects of perfusion volume andtemperature shifts on an alternating tangential flow perfusion with afed batch start-up as described above. All cultures were fed-batch startwith perfusion beginning on day 7. Perfusion flow rates moving fromthree-quarter working volumes to full volume or from full working volumeto three-quarter working volume were tested. A temperature shift from36° C. to 33° C. on Day 14 was also tested.

On day 0. CHO cells expressing a recombinant antibody were inoculatedinto 2 L production bioreactors at l×lO⁶ cells/mL in a working volume of1200 ml of a serum-free defined medium. Cultures were maintained at 36°C., DO at 30%. Prior to perfusion, glucose was fed to 7 g/L daily andduring perfusion glucose was maintained above 1 g/L. The culture wasmaintained for 20 days.

Cultures received bolus feeds of a concentrated serum-free defined feedmedia on day 4 (7.5% of initial working volume) and day 6 (10% initialworking volume). Perfusion began on Day 8. Perfusion rates are providedin Table 3. One culture from each group had a temperature shift from 36°C. to 33° C. on Day 15, the other cultures remained at 36° C. for theduration of the experiment

TABLE 3 Perfusion Rate Perfusion Rate Condition Day (Vol/day) Condition1 8-12 0.75 (n = 2) 12- 1.00 Condition 2 8-10 1.00 (n = 2) 10- 0.75Values are based on working volumes disclosed above

Temperature shift and perfusion rate did not impact viable cell density,see FIG. 3A. However, a temperature shift appears to help preserveviability at later time points in a culture. There appears to be a breakout between the temperatures shift conditions starting on Day 15 onward.The viability of the temperature shifted cultures dropped more slowlythan the cultures that remained at 36° C., see FIG. 3B. As for titer,three cultures showed very similar titers on Day 15 (17.1-17.9 g/L) aswell as on Day 20 (22-24 g/L), but one culture had a higher titer on Day15 (21.58 g/L) as well as on Day 20 (28.33 g/L) (see FIG. 3C). Neitherthe temperature nor perfusion rates appeared to have any impact on titerproduction, suggesting that cultures can be maintained with differentperfusion rates.

Example 4

This experiment was designed to investigate the effects of perfusionmedium asparagine concentrations and perfusion start conditions witheither L-asparagine limited or non-limited culture environments onviable cell density during the production phase.

On day 0, CHO cells expressing a recombinant antibody were inoculatedinto 2 L production bioreactors at l×l 0⁶ cells/mL in a working volumeof 1500 ml for both the batch and fed-batch start methods. Cultures weremaintained at 36° C., dissolved oxygen (DO) at 30%, agitation at 400RPM. Sparging was done using either a drilled pipe or a sinteredsparger. Glucose was maintained above 0 g/L and below 8 g/L.

Perfusion (alternating tangential flow) was started on day 3 (0.29Vol/day) for the batch start “non-asparagine-limited cultures” and onday 7 (0.48 Vol/day) for the fed-batch “asparagine-limited cultures”.The batch culture medium contained lOmM L-asparagine. Prior to startingperfusion the fed-batch cultures received bolus feeds of a concentratedserum-free defined feed media on days 3 and 6 (7% initial workingvolume) containing 113.4 mM L-asparagine. Perfusion medium asparagineconcentrations were either at a control concentration (17.3 mM Asn in aserum free defined perfusion medium) or a low concentration (5 mM Asn ina serum free defined perfusion medium). Perfusion was carried out asdescribed above. Perfusion rates are provided in Table 4.

TABLE 4 Perfusion Rates Perfusion Rate Condition Day (Vol/day) Day 3 3-4 0.29 Batch  4-7 0.48 Perfusion  7-9 0.48 Start  9-11 0.67 non-asparagine- limited culture 11-20 0.96 Day 7  7-9 0.48 Fed-Batch  9-110.67 Perfusion 11-20 0.96 Start asparagine- limited culture

During the culture run, daily samples were taken to assess the culture.Viable cell density (VCD) and viability were determined using Vi-Cell(Beckman Coulter, Brea, Calif.). Titer was measured by HPLC analysis.All cultures were maintained at 36.0° C.

Reduction in cell growth and improved productivity was achieved duringthe production phase by limiting asparagine in the culture medium. Onday 15, the maximum viable cell density was about 17.0×10⁶ viablecells/mL for the fed-batch start cultures with low asparagine (FIG. 4A).Control level asparagine cultures reached viable cell densitiesexceeding 40×10⁶ viable cells/mL (>30% packed cell volume). Viability ofthe low asparagine fed-batch culture was 67.1% while the batch cultureviability was 55.1% and the control was 69% (FIG. 4B). The packed cellvolume adjusted titer of the low asparagine fed-batch culture was 17.0g/L (adjusted for packed cell volume) while the batch culture titer wasbetween 15.4 g/L (FIG. 4C). The controls had titers of 10.2 to 12.9 g/L(batch start) and 14.2 to 15.9 g/L (fed-batch start).

Maintaining asparagine levels at 5 mM or less during the productionresulted in growth-arrest, stimulated productivity and maintainedviability during the production phase.

Example: 5

This experiment compares media conditions during perfusion. In this 2 Lbioreactor experiment, cells were inoculated into a chemically definedbatch medium at a working volume of 1.5 L, cultured for 3 days and thenperfused for 12 days using a chemically defined perfusion mediumcontaining either 17.3 mM L-asparagine and 4.6 mM L-glutamine or 5 mML-asparagine and 10 mM L-glutamine. Perfusion was accomplished using analternating tangential flow perfusion and filtration system (RefineTechnologies, Hanover, N.J.) with a 30 kDa hollow fiber filter (GEHealthcare, Little Chalfont, UK). Perfusion was started on day 3 at arate of 0.3 culture volumes per day. The rate of perfusion was increasedon days 4, 9, and 11 as indicated in Table 6 below. Cultures weremaintained at 36° C., DO at 30%, pH at 7.0, and agitation at 400 rpm.

During the culture run, daily samples were taken to assess the culture.Viable cell density (VCD) and viability were determined using Vi-Cell(Beckman Coulter, Brea, Calif.). Titer was measured by HPLC analysis.Packed cell volume was determined using VoluPAC (Sartorius, Goettingen,Germany).

TABLE 5 Perfusion rate schedule Day Perfusion Rate (Vol/day)  3 0.30  40.50  9 0.67 11 0.96

Asparagine limitation resulted in the accumulation of fewer cells andimproved productivity. Cultures perfused with media containing 5 mMasparagine reached a maximum VCD of 8.16×10⁷-8.54×10⁷ cells/mL whilecultures perfused with media containing 17.3 mM asparagine reached11.9×10⁷-12.2×10⁷ cells/mL (FIG. 5A). Although the cultures in 17.3 mMasparagine had more cells, the cultures in 5 mM asparagine made moreproduct. Cultures perfused with media containing 17.3 mM asparagine made6.89-7.18 g/L (4.33-4.67 g/L packed cell volume adjusted) compared to7.59-8.15 g/L (5.01-5.38 g/L packed cell volume adjusted) for culturesperfused with media containing 5 mM asparagine (FIGS. 5B and 5D). Thefinal packed cell volume (PCV) of the 5 mM asparagine cultures trendedslightly lower than the 17.3 mM asparagine cultures (FIG. 5C) and therewas no difference in culture viability (FIG. 5E).

Interestingly, in this example increasing the concentration of glutamineby more than two-fold in the low-asparagine condition (4.6 mM versus 10mM glutamine) did not interfere with the ability of the low-asparaginemedium to arrest the growth of the culture.

Example 6

This example compares the performance of a clonal, antibody-expressingCHO cell line cultured in an ATF perfusion process using asparaginelimitation to control growth at bench and pilot scales. The bench-scalemodel utilized 2 L bioreactors and the pilot scale was 500 L. At benchscale, cells were inoculated into a chemically defined batch medium at aworking volume of 1.5 L and at pilot scale the working volume was about378 L. Cells were cultured for 3 days in the batch medium and thenperfused for 12 days using a chemically defined perfusion mediumcontaining 5 mM L-asparagine and 10 mM L-glutamine. Perfusion wasaccomplished using an alternating tangential flow perfusion andfiltration system (Refine Technologies, Hanover, N.J.) with a 30 kDahollow fiber filter (GE Healthcare, Little Chalfont, UK). Perfusion wasstarted on day 3 at a rate of 0.3 culture volumes per day. The rate ofperfusion was increased on days 4, 9, and 11 as indicated in Table 7below. Cultures were maintained at 36° C. 30% DO, and pH 6.9.

During the culture run, daily samples were taken to assess the culture.Viable cell density (VCD) and viability were determined bench scaleusing Vi-Cell (Beckman Coulter, Brea, Calif.) and at pilot scale using aCEDEX (Roche Applied Science, Indianapolis, Ind.). Titer was measured byHPLC analysis. Packed cell volume was determined using VoluPAC(Sartorius, Goettingen, Germany).

TABLE 6 Perfusion rate schedule Day Perfusion Rate (Vol/day)  3 0.30  40.50  9 0.67 11 0.96

Data from four bench scale cultures and two pilot scale cultures isprovided. VCD curves were similar at both scales, growth control wasachieved (FIG. 6A), and the total cell mass (packed cell volume) wasmaintained below 30% at both scales (FIG. 6C). Although the VCD reacheda plateau around day 10 or day 11, the packed cell volume continued toincrease until about day 13 or 14 (FIG. 6C). Productivity was alsosimilar between scales. Cultures perfused with media containing 5 mMasparagine made 14.2-15.7 g/L (10.7-11.4 g/L packed cell volumeadjusted) at 2 L bench scale compared to 15.0-17.3 g/L (10.6-12.8 g/Lpacked cell volume adjusted) at 500 L pilot scale (FIGS. 6B and 6D).Viability trended slightly lower at the pilot scale (FIG. 6E).

What is claimed is:
 1. A method of limiting a mammalian cell culture ata packed cell volume (PCV) of 35% or less, wherein said mammalian cellexpresses a recombinant protein, comprising: establishing a mammaliancell culture in a serum-free culture medium in a bioreactor byinoculating the bioreactor with at least 0.5×10⁶ to 3.0×10⁶ cells/mL,limiting the PCV of said culture by perfusion with a serum-freeperfusion medium having an L-asparagine concentration of 5 mM or less,maintaining the mammalian cells by perfusion with a serum-free perfusionmedium having an L-asparagine concentration of 5 mM or less, andmonitoring the cell culture, such that the PCV of the culture ismaintained at 35% or less.
 2. The method of claim 1, wherein theperfusion with a serum-free perfusion medium having an L-asparagineconcentration of 5 mM or less begins on or before day 3 of the culture.3. The method of claim 1, wherein perfusion begins at a time that isbetween day 5 and day 9 of the cell culture.
 4. The method of claim 1,wherein perfusion begins at a time that is between day 5 and day 7 ofthe cell culture.
 5. The method according to claim 1, wherein perfusionwith a serum-free perfusion medium having an L-asparagine concentrationof 5 mM or less takes place prior to a production phase.
 6. The methodof claim 1, wherein perfusion begins when the cells have reached aproduction phase.
 7. The method according to claim 1, wherein perfusionwith a serum-free perfusion medium having an L-asparagine concentrationof 5 mM or less takes place during a production phase.
 8. The method ofclaim 1, wherein the concentration of L-asparagine in the serum-freeperfusion medium is less than or equal to 4.0 mM.
 9. The method of claim1, wherein the concentration of L-asparagine in the serum-free perfusionmedium is less than or equal to 3.0 mM.
 10. The method of claim 1,wherein the concentration of L-asparagine in the serum-free perfusionmedium is less than or equal to 2.0 mM.
 11. The method of claim 1,wherein the concentration of L-asparagine in the serum-free perfusionmedium is less than or equal to 1.0 mM.
 12. The method of claim 1,wherein the concentration of L-asparagine in the serum-free perfusionmedium is equal to 0 mM.
 13. The method of claim 1, wherein theL-asparagine concentration of the cell culture medium is monitored. 14.The method of claim 1, wherein the PCV is maintained at 30% or less. 15.The method of claim 1, wherein the PCV is maintained at between 10% and35%.
 16. The method of claim 1, wherein the PCV is maintained at between20% and 35%.
 17. The method according to claim 1, further comprising atemperature shift, wherein the temperature is lowered from between 35°C. and 38° C. to between 30° C. and 34° C.
 18. The method according toclaim 17, wherein the temperature shift occurs at the transition betweena growth phase and a production phase.
 19. The method according to claim17, wherein the temperature shift occurs during a production phase. 20.The method of claim 1, further comprising a temperature shift, whereinthe growth phase occurs at a first temperature that is between 35° C.and 38° C. and the production phase occurs at a second temperature thatis between 30° C. and 34° C.
 21. The method according to claim 1,further comprising a temperature shift from 36° C. to 31° C.
 22. Themethod according to claim 1, further comprising a temperature shift from36° C. to 33° C.
 23. The method of claim 1, wherein the mammalian cellsare Chinese Hamster Ovary (CHO) cells.
 24. A method of limiting amammalian cell culture at a viable cell density (VCD) of 1×10⁸ cells/mLor less, wherein said mammalian cell expresses a recombinant protein,comprising: establishing a mammalian cell culture in a serum-freeculture medium in a bioreactor by inoculating the bioreactor with atleast 0.5×10⁶ to 3.0×10⁶ cells/mL, limiting the VCD of said culture byperfusion with a serum-free perfusion medium having an L-asparagineconcentration of 5 mM or less, maintaining the mammalian cells byperfusion with a serum-free perfusion medium having an L-asparagineconcentration of 5 mM or less, and monitoring the cell culture, suchthat the VCD of the culture is maintained at 1×10⁸ cells/mL or less. 25.The method of claim 24, wherein perfusion begins at a time that isbetween day 5 and day 9 of the cell culture.
 26. The method of claim 24,wherein perfusion begins at a time that is between on or about day 5 andto on or about day 7 of the cell culture.
 27. The method of claim 24,wherein perfusion with a serum-free perfusion medium having anL-asparagine concentration of 5 mM or less takes place prior to aproduction phase.
 28. The method of claim 24, wherein perfusion beginswhen the cells have reached a production phase.
 29. The method of claim24, wherein perfusion with a serum-free perfusion medium having anL-asparagine concentration of 5 mM or less takes place during aproduction phase.
 30. The method of claim 24, wherein the concentrationof L-asparagine in the serum-free perfusion medium is less than or equalto 4.0 mM.
 31. The method of claim 24, wherein the concentration ofL-asparagine in the serum-free perfusion medium is less than or equal to3.0 mM.
 32. The method of claim 24, wherein the concentration ofL-asparagine in the serum-free perfusion medium is less than or equal to2.0 mM.
 33. The method of claim 24, wherein the concentration ofL-asparagine in the serum-free perfusion medium is less than or equal to1.0 mM.
 34. The method of claim 24, wherein the concentration ofL-asparagine in the serum-free perfusion medium is equal to 0 mM. 35.The method of claim 24, wherein the L-asparagine concentration of thecell culture medium is monitored.
 36. The method of claim 24, whereinthe VCD is maintained at 90×10⁶ cells/mL or less.
 37. The method ofclaim 24, wherein the VCD is maintained at between 40×10⁶ cells/mL and100×10⁶ cells/mL.
 38. The method of claim 24, further comprising atemperature shift, wherein the temperature is lowered from between 35°C. and 38° C. to between 30° C. and 34° C.
 39. The method of claim 38,wherein the temperature shift occurs at the transition between a growthphase and a production phase.
 40. The method of claim 38, wherein thetemperature shift occurs during a production phase.
 41. The method ofclaim 24, further comprising a temperature shift, wherein the growthphase occurs at a first temperature that is between 35° C. and 38° C.and the production phase occurs at a second temperature that is between30° C. and 34° C.